1. Field of the Invention
This invention relates to four novel strains of Bacillus thuringiensis (the "BTS02617A strain", the "BTS02618A strain", the "BTS02654B strain" and the "BTS02652E strain"), each of which produces crystallized proteins (the "BTS02617A crystal proteins", the "BTS02618A crystal proteins", the "BTS02654B crystal proteins" and the "BTS02652E crystal proteins", respectively) which are packaged in crystals (the "BTS02617A crystals", the "BTS02618A crystals", the "BTS02654B crystal" and the "BTS02652E crystals", respectively) during sporulation. The BTS02617A, BTS02618A, BTS02654B and BTS02652E strains were deposited under the provisions of the Budapest Treaty at the Belgian Coordinated Collections of Microorganisms--Collection Laboratorium voor Microbiologie Belgium ("BCCM-LMG"), R.U.G., K. Ledeganckstraat 35, B-9000 Gent.
This invention also relates to an insecticide composition that is active against Lepidoptera and that comprises the BTS02617A, BTS02618A, BTS02654B or BTS02652E strain, as such, or preferably the BTS02617A, BTS02618A, BTS02654B or BTS02652E crystals, crystal proteins or the active component(s) thereof as an active ingredient.
This invention further relates to a gene (the "bTS02618A gene"), which is present in the genome of the BTS02617A, BTS02618A, BTS02654B and BTS02652E strains and which encodes an insecticidal protein (the "BTS02618A protoxin") that is found in the BTS02617A, BTS02618A, BTS02654B and BTS02652E crystals. The BTS02618A protoxin is the protein that is produced by the BTS02617A, BTS02618A, BTS02654B and BTS02652E strains before being packaged into their respective BTS02617A, BTS02618A, BTS02654B and BTS02652E crystals.
This invention still further relates to a toxin (the "BTS02618A toxin") which can be obtained (e.g., by trypsin digestion) from the BTS02618A protoxin. The BTS02618A toxin is an insecticidally active protein which can be liberated from the BTS02617A crystals, the BTS02618A crystals, the BTS02654B crystals, and the BTS02652E crystals, which are produced by the BTS02617A strain, the BTS02618A strain, the BTS02654B strain and the BTS02652E strain, respectively. This toxin and its protoxin have a high activity against a wide range of lepidopteran insects, particularly against Noctuidae, especially against Spodoptera and Agrotis spp., but also against other important lepidopteran insects such as Pyralidae, particularly the European corn borer, Ostrinia nubilalis, Gelechiidae such as Phthorimaea operculella and Yponomeutidae such as Plutella xylostella. Furthermore, the BTS02618A protein is the first Bt protein with significant activity towards Agrotis segetum. This new characteristic of the BTS02618A protoxin and toxin ("(pro)toxin"), i.e., the combination of activity against different economically important Lepidopteran insect families such as Noctuidae, Yponomeutidae, Gelechiidae and Pyralidae, makes this (pro) toxin an ideally suited compound for combatting a wide range of insect pests by contacting these insects with the (pro)toxin, e.g., by spraying or by expressing the bTS02618A gene in plant-associated bacteria or in plants. The BTS02618A toxin is believed to represent the smallest portion of the BTS02618A protoxin which is insecticidally effective against Lepidoptera.
This invention also relates to transformed Bacillus thuringiensis strains, containing DNA sequences encoding a BTS02618A protein or variants thereof having substantially the same insecticidal activity.
This invention yet further relates to a chimeric gene that can be used to transform a plant cell and that contains the following operably linked DNA fragments:
1) a part of the bTS02618A gene (the "insecticidally effective bTS02618A gene part") encoding an insecticidally effective portion of the BTS02618A protoxin, preferably a truncated part of the bTS02618A gene (the "truncated bTS02618A gene") encoding just the BTS02618A toxin; PA1 2) a promoter suitable for transcription of the insecticidally effective bTS02618A gene part in a plant cell; and PA1 3) suitable 3' end transcript formation and polyadenylation signals for expressing the insecticidally effective bTS02618A gene part in a plant cell. This chimeric gene is hereinafter generally referred to as the "bTS02618A chimeric gene". PA1 1) a cell (the "transformed plant cell") of a plant, such as corn or cotton, the genome of which is transformed with the insecticidally effective bTS02618A gene part, preferably the bTS02618A chimeric gene; and PA1 2) a plant (the "transformed plant") which is regenerated from the transformed plant cell or is produced from the so-regenerated plant and their seeds, the genome of which contains the insecticidally effective bTS02618A gene part, preferably the bTS02618A chimeric gene, and which is resistant to Lepidoptera. PA1 1) a microbial organism, such as B. thuringiensis or Pseudomonas spp., the genome of which is transformed with all or part of the bTS02618A gene; and PA1 2) a microbial spore, containing a genome which is transformed with all or parts of the bTS02618A gene. PA1 1) a DNA sequence encoding an insecticidally effective portion of the BTS02618Aa protoxin, preferably a truncated part of the bTS02618Aa gene (the "truncated bTS02618Aa gene") encoding just the BTS02618Aa toxin; PA1 2) a promoter suitable for transcription of the insecticidally effective bTS02618Aa gene part in a plant cell; and PA1 3) suitable 3' end transcript formation and polyadenylation signals for expressing the insecticidally effective bTS02618Aa gene part in a plant cell. This chimeric gene is hereinafter generally referred to as the "bTS02618Aa chimeric gene". PA1 1) a cell (the "transformed plant cell") of a plant, such as corn or cotton, the genome of which is transformed with the insecticidally effective bTS02618Aa gene part, preferably the bTS02618Aa chimeric gene; and PA1 2) a plant (the "transformed plant") which is regenerated from the transformed plant cell or is produced from the so-regenerated plant and their seeds, the genome of which contains the insecticidally effective bTS02618Aa gene part, preferably the bTS0261Aa chimeric gene, and which is resistant to Lepidoptera. PA1 1) a microbial organism, such as B. thurinciensis or Pseudomonas spp., the genome of which is transformed with all or part of a DNA sequence encoding the BTS02618Aa protein; and PA1 2) a microbial spore, containing a genome which is transformed with all or part of the bTS02618Aa gene.
This invention also relates to:
This invention still further relates to:
Another embodiment of the present invention relates to artificially made bTS02618A genes which encode BTS02618A proteins, and to proteins which are more protease resistant than native Bt proteins, more preferably the native BTS02618A protein. A particular example of a protein that is more protease resistant is the BTS02618Aa protein. Furthermore, the present invention also relates to a DNA sequence encoding the BTS02618Aa protein.
Yet another embodiment of the present invention relates to a chimeric gene that can be used to transform a plant cell and that contains:
This invention further relates to:
This invention still further relates to:
Yet another embodiment of the present invention relates to insecticidal compositions that are active against Lepidoptera and that comprise a more protease resistant Bt protein, more particularly the BTS02618Aa protein or a variant thereof which has substantially the same insecticidal activity.
2. Description of the Prior Art
B. thurinciensis ("Bt") is a Gram-positive bacterium which produces endogenous crystals upon sporulation. The crystals are composed of proteins which are specifically toxic against insect larvae. These crystal proteins and corresponding genes have been classified based on their structure and insecticidal spectrum (Hofte and Whiteley, 1989). The four major classes are Lepidoptera-specific (cryI), Lepidoptera- and Diptera-specific (cryII), Coleoptera-specific (cryIII), and Diptera-specific (cryIV) genes.
The fact that conventional submerged fermentation techniques can be used to produce Bt spores on a large scale makes Bt bacteria commercially attractive as a source of insecticidal compositions.
Gene fragments from some Bt strains, encoding insecticidal proteins, have heretofore been identified and integrated into plant genomes in order to render the plants insect-resistant. However, obtaining expression of such Bt gene fragments in plants is not a straightforward process. In order to achieve optimal expression of an insecticidal protein in plant cells, it has been found necessary to engineer each Bt gene fragment in a specific way so that it encodes a part of a Bt protoxin that retains substantial toxicity against its target insects (European patent application ("EPA") 86/300,291.1 and 88/402,115.5; U.S. patent application Ser. No. 821,582, filed Jan. 22, 1986).